Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks

Abstract

The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ∼10 hr. Furthermore, repair of the DSBs tends to be error prone. Both classical and microhomology-mediated end joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci and indicates that Cas9-induced DSBs are repaired in an unusual manner.

Keywords: CRISPR-Cas; DNA double-strand break; genome editing; microhomology-mediated end-joining; non-homologous end-joining; repair kinetics.

Graphical abstract

Figure 1

Quantitative Analysis of Cas9-Induced DSBs (A) Proposed model of DSB repair based on stochastic transitions between intact, broken, and indel states. k_p and k_m are rate constants of perfect and mutagenic repair, respectively; k_c is the rate constant of cutting by Cas9. The latter depends on Cas9 activation and is therefore denoted as k_c(t). (B) ODEs describing the three reaction steps, with rate constants as parameters. (C) Outline of the experimental strategy; see main text. (D) Representative time course experiment, showing gradual accumulation of indels. A sigmoid curve was fitted to the data to determine the plateau level at late time points (dashed line), which reflects the transfection efficiency. (E) Relative proportions of intact (red) and indel (green) fractions at the LBR2 locus over time. The data points are normalized on to the total indel fraction to correct for the variation in transfection efficiency. Indel fraction in absence of Shield-1 is shown in gray. Average of 7 independent experiments is shown; error bars represent the SD, and the dashed lines show fitted sigmoid curves. (F) Western blot analysis of Cas9 presence as a function of time. Tubulin was used as loading control. (G) The intensities of Cas9 antibody signal were determined by densitometry from time points of 3 individual western blots and normalized to a sample incubated for 60 hr with Shield-1 (lanes labeled “K562#17+Shield-1” in F). An ODE fit was performed to determine the activity score of Cas9 in time.

Figure 2

Estimation of Cutting and Repair Rates (A–C) Representative time series traces of the intact (A), broken (B), and indel (C) fractions. Measured data (dots) are overlaid with the ODE model fit (solid lines). The percentage of intact and indel traces are relative to the total. The broken fraction is estimated by the model on the basis of the intact and indel measurements. τ indicates time of the largest amount of broken DNA. (D) Distributions of rate constants from 7 independent experiments (values indicate mean ± SD). (E) Estimate of the upper confidence bound of the proportion of perfect repair. Fitting residual errors are shown for various fixed k_p/(k_p+k_m) ratios (7 combined time series). Black dashed line marks the optimal fit; red dashed line marks the k_p/(k_p+k_m) ratio above which the fit becomes significantly worse than the optimal fit (p < 0.1; one-sided F-test). This corresponds to k_p/(k_p+k_m) = 0.28. For each k_p/(k_p+k_m) ratio, k_c,max and k_m were adjusted to produce the best fit. (F) Examples of model fitting with various combinations of k_p and k_m forced to values that would correspond to models of rapid cycling of cutting and perfect repair (dashed curves). Green dots are measured values; green curve shows unrestrained optimal fit.

Figure 3

Measurement of Broken Fraction (A) Schematic view of the ligation mediated (LM)-PCR assay to detect the broken ends after DSB induction. See text for explanation. (B) Representative agarose gel of the LM-PCR products of a time series. The expected product is 205 bp in size. (C) The broken fraction measured as band intensities (data from 8 LM-PCR experiments from 4 independent time series; values are a.u.; mean ± SD). Solid blue line shows an ODE curve fit to the LM-PCR data to determine τ (blue shading, mean ± SD). (D and E) Same as (B) and (C) but with primers 2 and 3 that do not span the break site. The expected product is 163 bp in size. Values are in a.u., mean ± SD, based on 7 PCR experiments from 3 independent time series.

Figure 4

Multiple Repair Pathways Active at One Locus (A and B) The spectrum of indels and their frequencies at the LBR2 locus at time point t = 60 hr in cells cultured without (A) or with (B) 1 μM NU7441. A representative experiment is shown. Light-blue bar, wild-type sequence; dark-blue bars, indels. (C) Frequencies of −7 and +1 indels in the presence (black, n = 7) and absence (gray, n = 4) of NU7441. All series are normalized to the total indel fraction. Asterisks indicate p values according to Student’s t test: ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005. (D) The −7 deletions consist of two types; red nucleotides mark the deleted DNA. Shaded nucleotides show possible models for microhomology-mediated repair. Percentages indicate the proportion of observed −7 sequence reads. (E) TIDE analysis of +1 insertion and −7 deletion indels after exposure of the cells to 10 Gy of IR at the time of Cas9 induction. Asterisks indicate p values according to Student’s t test: ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005.

Figure 5

MMEJ Has Slower Repair Kinetics Than C-NHEJ (A) Kinetic model of Cas9-induced DSB repair assuming that each type of indel is generated with a specific repair rate. (B) +1 insertion and −7 deletion accumulation in a representative time series. Dashed lines show a Gompertz sigmoid fit. (C) Same measurements as in (B) with the multi-indel ODE model fit (solid lines) for the +1 (top) and −7 (bottom) indels. (D) Cartoon representation of hypothesized time dependency of the k₊₁ and k₋₇ rates added to the fitted model in (E). (E) Time-dependent ODE model fit of the +1 insertion (top) and −7 deletion (bottom) as proposed in (D). (F) Rate constants without (black) and with (gray) NU7441. The total mutagenic repair rate is represented by k_m; rate constant for the +1 insertion is k₊₁ and rate constants for the −7 deletion are k_{−7,t = 0hr} and k_{−7,t = 60hr} at the start and end of the time series, respectively. n indicates the number of time series. Asterisks indicate p values according to Wilcoxon test: ^∗p < 0.05; ^∗∗p < 0.005; ^∗∗∗p < 0.0005. (G) Broken fraction measurements in presence of NU7441 of 2 independent time series, similar to Figures 3B and 3C.

Figure 6

ODE Modeling of DSBs at Additional Loci; Cas9 Remains Bound to Broken Ends (A–H) Time series of Cas9 cutting and repair at four loci (LBR2, A and B; LBR8, C and D; AAVS1, E and F; intergenic region on chromosome 11, G and H). (A, C, E, and G) Intact fraction abundance of all individual replicates is shown in small dots where each replicate has a unique color. The mean of each time point is shown as big red dots. Fitted models are shown as red curves. (B, D, F, and H) Predicted broken fraction curves. (I and J) In vitro digestion of DNA fragments by Cas9 and one of the sgRNAs targeting LBR (orange), AAVS1 (purple), or intergenic region on chromosome 11 (brown), respectively. DNA was incubated for either 2 hr (I) or overnight (J) in the absence (uncut) or presence (cut) of Cas9 and sgRNA (Cas9•sgRNA). In some samples, Cas9 was subsequently denatured by two different heat treatment protocols as indicated. The expected band for the intact DNA is marked by an arrowhead, and the expected digestion products are marked by asterisks.

Figure 7

DSB Repair Fidelity after Double Cutting (A) Repair products that may result from Cas9 in combination with two sgRNAs that target adjacent sequences. All products are amplified by PCR and detected by high-throughput sequencing. (B) Quantification of the various repair products 60 hr after Cas9 induction in the presence of sgRNA-LBR2 and one of 5 sgRNAs (labeled 5 through 9) that target a sequence within 110–300 bp from the LBR2 site. (C) Same as (B) but highlighting the relative proportions of perfect and imperfect junctions among the repaired excision events. (D) Relative proportions of indels at sgRNA-LBR2 target site (blue), indels at sgRNA-LBR8 target site (red), indels at both sgRNA-LBR2 and sgRNA-LBR8 target sites (green), excised DNA (yellow), and wild-type (black) fractions over time. The data points are normalized to the total mutation fraction to correct for the variation in transfection efficiency. Average of 2 independent experiments is shown; error bars represent the SD. Dashed lines show fitted sigmoid curves.